Hsin-Cheng Chiu

Polymer Vesicles Containing Small Vesicles within Interior Aqueous Compartments and pH-Responsive Transmembrane Channels†

Intermolecular packing of amphiphilic block copolymers into vesicles is of particular interest, owing to the fundamental importance of such systems as a new class of polymer assemblies with well-controlled structures and potential biomedical applications. Similar to conventional liposomes, polymer vesicles usually form a continuous bilayer structure primarily consisting of the hydrophobic blocks of copolymers, but they exhibit markedly enhanced stability and feasibility of incorporating functional groups in response to external stimuli. However, the major limitation of polymer vesicles as biofunctional containers arises from the lack of permeation pathway for hydrophilic cargoes owing to the requirement to maintain the architectural integrity. The vesicles obtained from block co-polypeptides are imparted responsive channels upon the pH-induced conformational change of a polypeptide block. Redox control of the permeability of multilayer microcapsules containing poly(ferrocenylsiliane) was reported. Incorporating channelforming proteins into the vesicle membranes while fully retaining the protein functions represents an important paradigm of equipping polymer vesicles with transmembrane channels. Thus, the transport mechanism, being either size-selective or substrate-specific, can be tailored by the pore proteins selected. It is also desirable to have versatile vesicular assemblies that contain small vesicles within the interior aqueous compartments in a manner similar to discrete organelles within eukaryotic cells, which perform diverse functions and are one of the feature differences from prokaryotic counterparts. Unfortunately, such assembly structural control has not yet been achieved. Herein, we show the first example of polymeric multivesicle assemblies similar to the architectural arrangement of eukaryotic cells, in which both the vesicle membranes are equipped with pH-responsive channels permeable for hydrophilic solutes (Scheme 1). Copolymers comprising acrylic acid (AAc) and acrylate of 1,2-distearoyl-rac-glycerol (distearin acrylate, DSA) were obtained from partial transesterification of poly(N-acryloxysuccinimide) (poly(NAS)) with distearin and then thorough hydrolysis of the unreacted NAS to AAc units. Polymer vesicles were prepared by a double emulsion technique in a water/oil/water (w1/o/w2) system, in which the copolymer was dissolved in the organic phase prior to emulsification. The experimental methods are described in detail in the Supporting Information. THF/CH3Cl solutions of varying ratios, depending on the target vesicle size, were employed as the organic phase. Either water or buffers in the pH range of 4.0–5.5 were used as both the inner (w1) and outer (w2) aqueous phases. The vesicles formed upon the evaporation of organic solvents in w1/o/w2 emulsions. However, the copolymers assembled into micelles above pH 5.5 and large precipitates below pH 4.0. The vesicles were obtained mainly from copolymer with an average molecular weight of 2.97 ; 10 gmol 1 and a composition of 9.1 mol% DSA, unless stated otherwise. Figure 1a confirms that the resultant assemblies are unilamellar vesicles. The laser scanning confocal microscopy (LSCM) image of polymer vesicles in aqueous suspensions was revealed by the fluorescence of Nile red associated with the vesicle membranes. The lyophilized vesicles can be observed by scanning electron microscopy (see the Supporting Information). The fact that such polymer colloids maintain their structural integrity when subjected to transition from the aqueous to dried state reflects their robust stability. Transmission electron microscopy (TEM) examination of the sectioned specimens (ca. 60–90nm thickness) of polymer vesicles indicates that the wall thickness was approximately 25 nm (Figure 1b). The vesicle size can be controlled by adjusting either the THF/CH3Cl ratio used during emulsification or the DSA content of copolymers to give vesicles with diameters ranging from 1 to 15 mm. For example, changing the DSA content of copolymers from 9.1 to 13.1 mol% increases the vesicle diameter by 3–4 mm on the average. In contrast, increasing the THF content of the THF/CH3Cl solution from 2 to 20% (v/v) reduces the vesicle size significantly (Figure 2) because of the increased miscibility with water and the resulting decreased interfacial tension of the polymer-containing oil droplets in the aqueous phase. When the ionization of AAc residues increases to some extent with increasing pH value, the vesicles become equipped with transmembrane channels that are permeable for hydrophilic solutes. Figure 3 shows that, while transport of calcein (a water-soluble fluorescence probe) across the membrane was prohibited at pH 5.0, the probe molecules freely diffused into the vesicular aqueous compartment when the external pH value was increased to 8.0. Calcein was then confined within the compartment simply by adjusting the [*] Prof. H.-C. Chiu, Y.-W. Lin, Y.-F. Huang, C.-K. Chuang Department of Chemical Engineering National Chung Hsing University Taichung 402 (Taiwan) Fax: (+886)4-2285-2636 E-mail: hcchiu@dragon.nchu.edu.tw

Synthesis and characterization of amphiphilic poly(ethylene glycol) graft copolymers and their potential application as drug carriers

Amphiphilic graft copolymers comprising monomeric units of stearyl methacrylate, methyl acrylate, acrylic acid and poly(ethylene glycol) acrylate were synthesized and their properties in aqueous systems characterized. The structures of these copolymers were analysed by Fourier transform infra-red and nuclear magnetic resonance spectroscopies while their molecular weights were estimated by static light scattering. The study of critical micelle concentrations and micellar sizes indicated that the formation of micelles is primarily determined by the hydrophobic/hydrophilic properties of these copolymers. Encapsulation of pyrene (as a drug model) into the micelles was found to be dependent on their stearyl methacrylate content. These copolymers also exhibited a sustained release pattern for pyrene in aqueous solutions and might indicate their future applications as potential drug delivery systems.

Synthesis and characterization of acrylic acid-containing dextran hydrogels

pH-Sensitive dextran hydrogels were prepared by free radical polymerization of methacrylate derivatized dextran, acrylic acid and N-t-butylacrylamide. Incorporation of acrylic acid in hydrogels was confirmed by Fourier transform infrared spectroscopy. The pH-dependent swelling of hydrogels was strongly influenced by the acrylic acid content, conjugation degree of methacrylate moiety with dextran and modified dextran concentration. Intermolecular polymerization that occurred to a greater extent with a lower degree of conjugation of methacrylate and/or higher concentration of modified dextran effectively increased the network density of hydrogels. An increase of acrylic acid reduced the enzymatic degradability of pre-swollen hydrogels by dextranase, although the increased equilibrium swelling was observed.

Dual stimuli-responsive polymeric hollow nanogels designed as carriers for intracellular triggered drug release.

Dual stimuli-responsive hollow nanogel spheres serving as an efficient intracellular drug delivery platform were obtained from the spontaneous coassociation of two graft copolymers into the vesicle architecture in aqueous phase. Both copolymers comprise acrylic acid (AAc) and 2-methacryloylethyl acrylate (MEA) units as the backbone and either poly(N-isopropylacrylamide) (PNIPAAm) alone or both PNIPAAm and monomethoxypoly(ethylene glycol) (mPEG) chain segments as the grafts. The assemblies were then subjected to covalent stabilization within vesicle walls with ester-containing cross-links by radical polymerization of MEA moieties, thereby leading to hollow nanogel particles. Taking the advantage of retaining a low quantity of payload within polymer layer-enclosed aqueous chambers through the entire loading process, doxorubicin (DOX) in the external bulk phase can be effectively transported into the gel membrane and bound therein via electrostatic interactions with ionized AAc residues and hydrogen-bond pairings with PNIPAAm grafts at pH 7.4. With the environmental pH being reduced (e.g., from 7.4 to 5.0) at 37 °C, the extensive disruption of AAc/DOX complexes due to the reduced ionization of AAc residues within the gel layer and the pronounced shrinkage of nanogels enable the rapid release of DOX species from drug-loaded hollow nanogels. By contrast, the drug liberation at 4 °C was severally restricted, particularly at pH 7.4 at which the DOX molecules remain strongly bound with ionized AAc residues and PNIPAAm grafts. The in vitro characterizations suggest that the DOX-loaded hollow nanogel particles after being internalized by HeLa cells via endocytosis can rapidly release the payload within acidic endosomes or lysosomes. This will then lead to significant drug accumulation in nuclei (within 1 h) and a cytotoxic effect comparable to free drug. This work demonstrates that the novel DOX-loaded hollow nanogel particles show great promise of therapeutic efficacy for potential anticancer treatment.

Superparamagnetic Hollow Hybrid Nanogels as a Potential Guidable Vehicle System of Stimuli-Mediated MR Imaging and Multiple Cancer Therapeutics

Hollow hybrid nanogels were prepared first by the coassembly of the citric acid-coated superparamagnetic iron oxide nanoparticles (SPIONs, 44 wt %) with the graft copolymer (56 wt %) comprising acrylic acid and 2-methacryloylethyl acrylate units as the backbone and poly(ethylene glycol) and poly(N-isopropylacrylamide) as the grafts in the aqueous phase of pH 3.0 in the hybrid vesicle structure, followed by in situ covalent stabilization via the photoinitiated polymerization of MEA residues within vesicles. The resultant hollow nanogels, though slightly swollen, satisfactorily retain their structural integrity while the medium pH is adjusted to 7.4. Confining SPION clusters to such a high level (44 wt %) within the pH-responsive thin gel layer remarkably enhances the transverse relaxivity (r2) and renders the MR imaging highly pH-tunable. For example, with the pH being adjusted from 4.0 to 7.4, the r2 value can be dramatically increased from 138.5 to 265.5 mM(-1) s(-1). The DOX-loaded hybrid nanogels also exhibit accelerated drug release in response to both pH reduction and temperature increase as a result of the substantial disruption of the interactions between drug molecules and copolymer components. With magnetic transport guidance toward the target and subsequent exposure to an alternating magnetic field, this DOX-loaded nanogel system possessing combined capabilities of hyperthermia and stimuli-triggered drug release showed superior in vitro cytotoxicity against HeLa cells as compared to the case with only free drug or hyperthermia alone. This work demonstrates that the hollow inorganic/organic hybrid nanogels hold great potential to serve as a multimodal theranostic vehicle functionalized with such desirable features as the guidable delivery of stimuli-mediated diagnostic imaging and hyperthermia/chemotherapies.

Indocyanine Green-Encapsulated Hybrid Polymeric Nanomicelles for Photothermal Cancer Therapy

Indocyanine green (ICG), an FDA approved medical near-infrared (NIR) imaging agent, has been extensively used in cancer theranosis. However, the limited aqueous photostability, rapid body clearance, and poor cellular uptake severely restrict its practical applications. For these problems to be overcome, ICG-encapsulated hybrid polymeric nanomicelles (PNMs) were developed in this work through coassociation of the amphiphilic diblock copolymer poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-b-PEG) and hydrophobic electrostatic complexes composed of ICG molecules and branched poly(ethylenimine) (PEI). The ICG-encapsulated hybrid PNMs featured a hydrophobic PLGA/ICG/PEI core stabilized by hydrophilic PEG shells. The encapsulation of electrostatic ICG/PEI complexes into the compact PLGA-rich core not only facilitated the ICG loading but also promoted its aqueous optical stability. The effects of the chain length of PEI in combination with ICG on the physiochemical properties of PNMs and their drug leakage were also investigated. PEI(10k) (10 kDa) could form highly robust and dense complexes with ICG, and thus prominently reduced ICG outflow from the PNMs. The results of in vitro cellular uptake and cytotoxicity studies revealed that the ICG/PEI(10k)-loaded PNMs significantly promoted cellular uptake of ICG by HeLa cells due to their near-neutral surface, and thereby augmented the NIR-triggered hyperthermia effect in destroying cancer cells. These findings strongly indicate that the ICG/PEI10k-loaded PNMs have significant potential for attaining effective cancer imaging and photothermal therapy.

Thermally Induced Polymeric Assemblies from the PAAc-Based Copolymer Containing Both PNIPAAm and mPEG Grafts in Water

Graft copolymer comprising acrylic acid (AAc) units as the backbone and poly(N-isopropylacrylamide) (PNIPAAm) and monomethoxy poly(ethylene glycol) (mPEG) as the grafts undergoes phase transition and supramolecular assembly into colloidal particles in water upon the thermally induced hydrophobic association. The structural characteristics of the polymeric assemblies made from the graft copolymer in water are strongly dependent on the copolymer concentration and the way that the copolymer solution is subjected to heating from 25 degrees C to the phase transition region (occurring in the range 30 approximately 35 degrees C). The resultant assemblies are characterized by forming hydrophobic PNIPAAm regions with the multicore architecture and intercore connections. Interesting enough, these colloidal systems obtained from the copolymer solutions at different concentrations (10.0 and 1.0 mg/mL) and heating methods (fast and slow heating) exhibit very different structural responses when subjected to further temperature increase (from 30 approximately 35 to 60 degrees C). The mutual interactions among the components (PAAc backbone and PNIPAAm and mPEG grafts) of the copolymer were shown to play a crucial role in the evolution of the ultimate assembly structure. A molecular packing model was proposed to illustrate the mechanisms of the thermally induced structural transformation processes for the amphiphilic graft copolymer in water.